CN103725697B - The surface protein FnBPA genetic fragment of the staphylococcus aureus of chemosynthesis and expression, application - Google Patents
The surface protein FnBPA genetic fragment of the staphylococcus aureus of chemosynthesis and expression, application Download PDFInfo
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Abstract
The staphylococcus aureus surface albumen FnBPA genetic fragment of chemosynthesis of the present invention and expression thereof, application relate to technique for gene engineering, antibody and test kit field.The present invention is to be analyzed by computer, filter out the strong antigen epi-position in staphylococcus aureus surface albumen FnBPA, 745th amino acids is to the 877th amino acids, totally 133 aminoacid, select the codon that prokaryote is all had a preference for, the brand-new gene order of chemosynthesis epitope, utilizes technique for gene engineering, expresses this genetic fragment, prepares the strong antigen epitope Fragments of staphylococcus aureus surface albumen FnBPA.The strong antigen epitope Fragments of the staphylococcus aureus surface albumen FnBPA expressed, can be used for the detection of Staphylococcus aureus antibody and prepares monoclonal antibody and the multi-resistance etc. of anti-Staphylococcus aureus for immunity.
Description
Technical field
The present invention is the surface protein FnBPA partial gene fragments of the staphylococcus aureus of chemosynthesis, utilizes gene
Engineering, prepares the surface protein FnBPA of recombination staphylococcus aureus.Analyzed by computer, filter out containing strong antigen
The FnBPA fragment of epi-position, selects the codon of prokaryote preference, the gene order that chemosynthesis is brand-new, utilizes genetic engineering
Technological expression, the albumen of expression can be used for vaccine and the foundation etc. of staphylococcus aureus method for quick, the present invention relates to
Technique for gene engineering and diagnostic reagent field.
Background technology
Staphylococcus aureus is a kind of conditioned pathogen that can cause human and animal's disease.It is posted as normal flora
It is born in human skin and mucomembranous surface, accounts for the major part of hospital infection and Community Acquired Infections.S. aureus L-forms can cause skin and
Soft tissue infection, and life-threatening transitivity complication, such as pneumonia, bacteremia, endocarditis, suppurative arthritis and bone
Marrow is scorching, and it extensively retains in hospital, causes the host of immune system injury and surgical patient to infect, and retains in medical apparatus and instruments.
Staphylococcus aureus is ubiquitous in nature, all can find in the Excreta of empty gas and water, dust and humans and animals, because of
This, the food such as milk, meat, egg, fish and goods thereof is a lot of by its chance polluted.Staphylococcus aureus is also health organ at different levels
One of pathogen of emphasis detection.Therefore, the method for quick setting up staphylococcus aureus is extremely important.
The method step of conventional identification and separation staphylococcus aureus is more, and has certain limitation.First
According to detected pathogenic bacterium type, sample is carried out enrichment culture, its qualification result typically can only qualitative can not be quantitative, and
And required time is long, speed is slow, and general 3～4 days ability estimation result, precise Identification needs 5～7 days.Most of immunology skills
The experiment of art such as fluorescent-labeled antibody, enzyme linked immune assay (ELISA), radioimmunoassay test etc. are all used widely, but
Needed for them relatively costly, time-consuming and sample is carried out the process of complexity, limit its extensive application in life.Set up one
The detection method of the staphylococcus aureus simply, quickly, being suitable for scene application has important meaning.
Extracellular binding matrix albumen (ECMBP) that staphylococcus aureus produces, including FnBP, ClfA and Ebp etc.,
They are the protein ingredient of bacterium surface, have similar structure.FnbpA is the one of fibronectin binding protein (Fnbp)
Kind, it is the protein component of bacterium surface.By the double PCR of S. atreus clinical separation strain Fnbp is detected, send out
There is HpaA gene FnbpA in the bacterial strain of existing more than 95% and FnbpB gene only has 10%, and this research selects FnBPA as research
Object.
By computer software analysis, filter out the specific surfaces albumen FnPFA epitope of staphylococcus aureus
Enrichment region, selects the codon optimised genes sequence of prokaryote preference, the gene order that chemosynthesis is brand-new, utilizes gene work
Journey technological expression.The albumen expressed has preferable antigenicity and specificity, can be used for monoclonal antibody and polyclonal antibody
Preparation, the foundation for staphylococcus aureus method for quick lays the foundation.
Summary of the invention
The present invention is the brand-new genetic fragment of the staphylococcus aureus surface albumen FnBPA of chemosynthesis, utilizes gene
Engineering, prepares the Partial Fragment of the FnBPA of recombination staphylococcus aureus.Analyzed by computer, filter out containing strong anti-
The Partial Fragment of the staphylococcus aureus surface albumen FnBPA of former epi-position, the 745th amino acids the-the 877 amino acids,
Totally 133 aminoacid, selects the codon of prokaryote preference, the gene order that chemosynthesis is brand-new, utilizes genetic engineering skill
Art expresses this gene in e. coli bl21.The albumen expressed can be used for the preparation of monoclonal antibody and polyclonal antibody, for
The foundation of detection methods of staphylococcus aureus is laid a good foundation.
133 amino acid whose genetic fragments of the staphylococcus aureus surface albumen FnBPA of chemosynthesis and expression thereof,
Application takes following steps to implement:
1. the screening of staphylococcus aureus surface albumen FnBPA epitope and the chemosynthesis of genetic fragment thereof:
Utilize the softwares such as ANTHEWIN, DNAStar, analyzed the full amino of S. aureus L-forms surface protein FnBPA by computer
Acid sequence, sends out the N end (the 745th amino acids～the 877th amino acids) of S. aureus L-forms surface protein FnBPA containing stronger
Antigenic determinant.Select the codon of prokaryote preference, the gene order that chemosynthesis is brand-new, and add at 5' end
BamH I restriction enzyme site (lower setting-out part), adds termination codon (TGA) and Xho I restriction enzyme site (lower setting-out at 3' end
Part), make this genetic fragment be prone to be cloned in BamH I and the Xho I restriction enzyme site of plasmid pGEX4T-2.
Epitope aminoacid sequence in the staphylococcus aureus surface albumen FnBPA of screening (the 745th aa-the
877 aa):
Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Glu Asp Lys
Pro Lys Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln
Ile His Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys
Pro Lys Tyr Glu His Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His
Ile His Gly Phe Asn Lys His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys
Pro Ser Tyr Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro
Lys Val Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro
Ile Val
The DNA sequence (414bp) of the staphylococcus aureus surface albumen FnBPA antigen epitope genes of chemosynthesis as
Under:
GGATCC GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA
GAT AAG CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC
CCG CAG ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA
GAT AAG CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT
CCG CAT ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG
GAT AAA CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG
CTG CCG AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG
CCG CCG ATT GTT TGACTCGAG
2. the structure of expression staphylococcus aureus FnBPA fragment recombiant plasmid:
Extract plasmid pGEX4T-2, with BamH I and Xho I double digestion, after the agarose gel electrophoresis of 1.0%, reclaim enzyme
The plasmid large fragment cut, is dissolved in deionized water.The Staphylococcus aureus of same BamHI and Xho I double digestion chemosynthesis
Bacterium FnBPA fragment, reclaims purpose fragment, is dissolved in deionized water after the agarose gel electrophoresis of 1.0%.
Take the DNA fragmentation after the above two enzyme action of equimolar concentration, with T4 DNA ligase 16 in same centrifuge tube
DEG C overnight connect, make BamH I and Xho I position that staphylococcus aureus FnBPA genetic fragment is inserted in vector pGEX 4T-2
Between point, consistent with the start codon translation framework on carrier, express a fusion protein.
3. the screening of recombiant plasmid and qualification:
By recombinant plasmid transformed e. coli bl21 (DE3), coating, containing ampicillin (100 μ g/ml) LB flat board, puts 37
DEG C overnight.Next day, random picking converted bacterium colony, was seeded to the examination containing 4ml LB culture medium (containing ampicillin 100 μ g/ml)
Shaking in pipe, extracts recombiant plasmid, verifies with BamH I and Xho I double digestion, and the agarose gel electrophoresis result of 1.0% shows,
Cut the purpose fragment of about 414bp.Plasmid containing exogenous gene carries out DNA sequencing analysis, and sequencing result confirms restructuring matter
Grain is containing staphylococcus aureus FnBPA genetic fragment, and sequence is the most correct:
GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT AAG
CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG CAG
ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT AAG
CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG CAT
ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT AAA
CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG CCG
AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG CCG
ATT GTT
The expression of recombinant plasmid staphylococcus aureus FnBPA genetic fragment (133 aminoacid) built, melts at its N end
Having closed 226 aminoacid on carrier, 359 aminoacid of total length, its aminoacid sequence is as follows:
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr
Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp
Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu
Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser
Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys
Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln
Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln
Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly Ser
Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Glu Asp Lys Pro Lys
Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln Ile His
Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys Pro Lys
Tyr Glu His Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His Ile His
Gly Phe Asn Lys His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys Pro Ser
Tyr Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro Lys Val
Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro Ile Val
4. the Screening and Identification of expressed fusion protein engineering bacteria:
By the positive transformant containing recombiant plasmid, it is seeded to containing 4ml LB culture medium (containing ampicillin 100 μ g/ml)
Test tube in, 37 DEG C of shaken cultivation 4h, preserve strain.Remaining bacterium solution adds IPTG to final concentration 0.2mmol/L, continues vibration training
Supporting induction 4h, centrifugal thalline of collecting carries out SDS-PAGE detection, and recon expresses the golden yellow Portugal that relative molecular weight is 42 kDa
Grape coccus FnBPA albumen, expression is 20%, and compares bacterium pGEX4T-2 without this protein band.
5. the purification of expression staphylococcus aureus FnBPA albumen:
1) ultrasonic degradation of S. aureus L-forms FnBPA protein engineering bacterium is expressed
By engineering bacteria 8000 rpm of abduction delivering fusion protein, 4 DEG C, centrifugal 20 mins, thalline is resuspended in original fluid
In the lysate of 1/10 volume, lysate is 20mmol/L PB pH8.0,10 mmol/L EDTA, 1mmol/L DTT, 5% sweet
Oil, ice-bath ultrasonic breaks bacterium 75 times, 8000 rpm, 4 DEG C, centrifugal 20 mins, abandons precipitation, collects supernatant.Under the supernatant collected is used for
The affinitive layer purification of one step.
2) purification of staphylococcus aureus FnBPA albumen is expressed
Supernatant solution adds counter-balanced High-Affinity GSH Resin 10ml, and 4 DEG C combine overnight, and loading is received
Collection penetrates liquid.Washing pillar with 1 × PBS of ten times of bed volumes, then with the GSH eluent of 50ml high concentration, eluent is
50 mmol/L Tris-HCl pH8.0,10 mmol/L GSH, in three times eluting destination proteins, be the golden yellow Portugal of purification
Grape coccus FnBPA protein fragments.
6. the staphylococcus aureus FnBPA protein fragments expressed is for S. aureus vaccines.
7. the staphylococcus aureus FnBPA protein fragments will expressed, for immunity Balb/C mice, prepares specificity
Monoclonal antibody.
8. the staphylococcus aureus FnBPA protein fragments will expressed, for immunity new zealand white rabbit, prepares many grams
Grand antibodies Antibodies.
9. monoclonal antibody and the multi-resistance of preparation are assembled colloid gold reagent bar.
The present invention compared with prior art has the advantage that
The staphylococcus aureus surface albumen FnBPA fragment that we express, has a more advantages:
1. current, lack S. aureus vaccines.Vaccination can lifting body internal specific antibody titer, simultaneously the most also
Increase Cellular Immunity power, have synergism with antibiosis, and selection pressure produced by antibiotic can be reduced, delay drug resistance
The generation of bacterium.But owing to S. aureus vaccines is inactivation whole-bacterial-vaccine, in addition to protective immunity is former, possibly together with the most not
Relevant and poisonous composition, toxicity is relatively big, makes clinical practice limited.Researchers are also being actively working to new generation vaccine
Development.Surface protein FnBPA exists in the staphylococcus aureus of more than 95%, and we have selected its strong antigen epi-position, utilizes
Technique for gene engineering expresses preparation, lays the foundation for developing recombinant vaccine.Recombinant vaccine safety, low cost.
2, method that is current, that lack fast detecting Staphylococcus aureus.We select staphylococcus aureus surface egg
White FnBPA epitope enrichment region, immune animal, prepare specific antibody, assemble colloid gold reagent bar, improve golden yellow Fructus Vitis viniferae
The recall rate of coccus.
3., according to the staphylococcus aureus FnBPA fragment amino acid sequence filtered out, select the close of prokaryote preference
Numeral, the gene order that chemosynthesis is brand-new, this gene suitably high expressed in prokaryotic cell.
4. the engineering bacteria of the expression staphylococcus aureus surface albumen FnBPA built, expression is up to tropina
More than 20%, and express be soluble protein, it is easy to purification, this albumen can be prepared by large-scale purification.
Accompanying drawing explanation
Below with reference to accompanying drawing, the invention will be further described:
Fig. 1 is the molecular biology software full length amino acid sequence antigen to staphylococcus aureus surface albumen FnBPA
Epitope analysis result.Result shows, at N end from the 745th amino acids to the 877th amino acids, containing strong hydrophilic antigen
Epi-position, the i.e. position of arrows in figure.
Fig. 2 is the construction of recombinant plasmid flow chart expressing staphylococcus aureus surface albumen FnBPA.
Fig. 3 is the double digestion figure detecting recombiant plasmid with the agarose gel of 1.0%.M:DNA marker DL5000,1:
Recombiant plasmid pGEX4T-2-fnbpa cuts the purpose fragment of about 414bp, i.e. arrow mark in figure through BamH I and Xho I double digestion
The position shown.
Fig. 4 is the SDS-PAGE analysis result expressing staphylococcus aureus surface albumen FnBPA recombinant bacterium.
M: albumen marker (Quan Shijin)；C: comparison bacterium plasmid Han pGEX4T-2；1,2: recombinant bacterium, two equal tables of recon
Reach the fusion protein that relative molecular weight is about 42kDa, the i.e. position of arrows in figure.
Fig. 5 is to express staphylococcus aureus surface albumen FnBPA SDS-PAGE analysis result after purification.M: albumen
Marker (Quan Shijin)；1:High-Affinity GST Resin affinity column staphylococcus aureus surface after purification
Albumen FnBPA, OD280=1.4, concentration about 1.2mg/m；2:BSA(1mg/ml).
Fig. 6 is the result of ELISA detection rabbit anti-serum.The staphylococcus aureus surface albumen FnBPA immunity expressed is new
The blue White Rabbit in west prepares antiserum, and serum titer reaches 1:400000.
Detailed description of the invention
The detailed description of embodiment of the present invention:
Analysis, gene chemical synthesis and the expression of staphylococcus aureus surface albumen FnBPA epitope
Analyzed the full length amino acid sequence of staphylococcus aureus surface albumen FnBPA by computer, filter out golden yellow
Strong antigen epi-position in color staphylococcal surface protein FnBPA, selects the codon of prokaryote preference, translates into corresponding core
Nucleotide sequence, the gene order that chemosynthesis staphylococcus aureus surface albumen FnBPA strong antigen epi-position is brand-new.By gene sheet
Between BamH I and Xho I site that section is cloned in plasmid pGEX4T-2, with the translation framework one of the start codon on carrier
Cause, express a fusion protein.By recombinant plasmid transformed e. coli bl21 (DE3), it is golden yellow that screening obtains high efficient expression
The engineering bacteria of staphylococcal surface protein FnBPA, the staphylococcus aureus surface albumen FnBPA of expression accounts for tropina total amount
20%, and solvable.
Materials and methods
1. strain and plasmid: e. coli bl21 (DE3) and expression vector pGEX4T-2 are Nanjing Military Command's military medicine
Institute medicine bioengineering is preserved.
2. molecular biology reagents: restricted enzyme BamH I, Xho I and T4 DNA ligase are TaKaRa company
Product.Plasmid purification kit is Promega Products.DTT and IPTG is BIOSHARP Products.Other reagent is
Import or domestic analytical pure.
3. the synthesis of genetic fragment: helped synthesis by Nanjing Genscript Biotechnology Co., Ltd..
4. the enzyme action of gene clone method: DNA, connection；The extraction of plasmid, conversion；The SDS-PAGE analysis etc. of albumen
General molecular cloning method is carried out according to a conventional method.Other test kit by specification operates.
5. DNA sequence analysis: with Promega company plasmid purification kit plasmid purification, with the full-automatic sequenator of DNA
Order-checking.
Result
1. staphylococcus aureus surface albumen FnBPA epitope screening and the synthesis of genetic fragment:
Utilize ANTHEWIN, DNAStar equimolecular biological software, analyze staphylococcus aureus surface by computer
The full length amino acid sequence (GeneBank, ACCESSION:P14738) of albumen FnBPA, filters out staphylococcus aureus table
Strong antigen epi-position in the albumen FnBPA of face, i.e. from the 745th amino acids to the 877th amino acids, is shown in Fig. 1, its aminoacid sequence
As follows:
Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Glu Asp Lys
Pro Lys Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln
Ile His Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys
Pro Lys Tyr Glu His Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His
Ile His Gly Phe Asn Lys His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys
Pro Ser Tyr Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro
Lys Val Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro
Ile Val
The epitope aminoacid sequence in staphylococcus aureus surface albumen FnBPA according to screening, selects protokaryon
The codon of biological preference, the gene order that chemosynthesis is brand-new, and add BamH I restriction enzyme site (lower setting-out at 5' end
Part), increase termination codon (TGA) and XhoI I restriction enzyme site (lower setting-out part) at 3' end, make this genetic fragment be prone to
In the BamH I being cloned in plasmid pGEX4T-2 and Xho I restriction enzyme site.Chemosynthesis containing staphylococcus aureus surface
The DNA sequence (414bp) of albumen FnBPA antigen epitope genes is as follows:
GGATCC GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA
GAT AAG CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC
CCG CAG ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA
GAT AAG CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT
CCG CAT ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG
GAT AAA CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG
CTG CCG AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG
CCG CCG ATT GTT TGACTCGAG
2. the structure of expression staphylococcus aureus surface albumen FnBPA fragment recombiant plasmid:
Extract plasmid pGEX4T-2, with BamH I and Xho I double digestion, after the agarose gel electrophoresis of 1.0%, reclaim enzyme
The plasmid large fragment cut, is dissolved in deionized water.Same BamH I and the genetic fragment of Xho I double digestion chemosynthesis used, electricity
After swimming is reclaimed, it is dissolved in deionized water.
Take DNA fragmentation after the above two enzyme action of equimolar concentration, with T4 DNA ligase 16 in same centrifuge tube
DEG C, overnight connect, make the BamH I that staphylococcus aureus surface albumen FnBPA genetic fragment is inserted in vector pGEX 4T-2
And between Xho I site, consistent with the start codon translation framework on carrier, express a fusion protein, specifically build stream
Journey, is shown in Fig. 2.
3. the qualification of recombiant plasmid:
Converted product, to e. coli bl21 (DE3), is coated with the benzyl penicillium sp Han ammonia by the recombinant plasmid transformed upper step connected
On the solid LB media of element (100 g/ml), put 37 DEG C of overnight incubation.Next day, 2 transformant bacterium colonies of random choose, marked respectively
It is designated as 1, No. 2, chooses the comparison bacterium that 1 empty plasmid pGEX4T-2 converts simultaneously, be labeled as C, be inoculated into respectively and train containing 4ml liquid LB
Support in the test tube of base (containing ampicillin 100 g/ml), put 37 DEG C of shaken cultivation 5h, extract recombiant plasmid.With BamH I and
Xho I double digestion, detects with the agarose gel of 1.0%.Recombiant plasmid cuts out the genes of interest fragment of 414bp, and contains
The comparison bacterium of plasmid pGEX4T-2 does not cut out this genetic fragment, sees Fig. 3.Tentative confirmation, transformant contains Staphylococcus aureus
The genetic fragment of bacterium surface protein FnBPA.
Extracting the plasmid of recon, send Nanjing Genscript Biotechnology Co., Ltd. to check order, DNA sequence analysis confirms, weight
Group plasmid contains the staphylococcus aureus surface albumen FnBPA genetic fragment of synthesis, and sequence is the most correct:
GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT AAG
CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG CAG
ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT AAG
CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG CAT
ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT AAA
CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG CCG
AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG CCG
ATT GTT TGA
Expression of recombinant plasmid staphylococcus aureus surface albumen FnBPA fragment (133 aminoacid) built, at its N
End has merged 226 aminoacid on carrier, 359 aminoacid of total length, and its aminoacid sequence is as follows:
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr
Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp
Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu
Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser
Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys
Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys
Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln
Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln
Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly Ser
Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Glu Asp Lys Pro Lys
Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln Ile His
Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys Pro Lys
Tyr Glu His Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His Ile His
Gly Phe Asn Lys His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys Pro Ser
Tyr Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro Lys Val
Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro Ile Val
4. the Screening and Identification of expression staphylococcus aureus surface albumen FnBPA engineering bacteria:
Comparison bacterium positive transformant containing recombiant plasmid and 1 empty plasmid pGEX4T-2 converted, is seeded to containing 4ml
In the test tube of LB culture medium (containing ampicillin 100 g/ml), 37 DEG C of shaken cultivation 5h, after preserving strain, add final concentration extremely
The IPTG of 0.2mmol/L induces, and 25 DEG C are continued shaken cultivation 4h, and centrifugal thalline of collecting carries out SDS-PAGE detection, restructuring
Sublist reaches the staphylococcus aureus surface albumen FnBPA that relative molecular weight is about 42kDa, and expression is 20%, and compare bacterium without
This protein band, is shown in Fig. 4.
Express the purification of staphylococcus aureus surface albumen FnBPA
According to the aminoacid sequence of expression staphylococcus aureus surface albumen FnBPA, analyze its physicochemical property, determine suitable
When purification process.We expressed staphylococcus aureus surface albumen FnBPA merges the GST albumen on carrier,
Gst fusion protein have expressed glutathione S when expressing simultaneously and turns enzyme, can easily separate, therefore with GST agarose gel FF
Determine to use affinity chromatography, be purified with High-Affinity GST Resin.Specifically comprise the following steps that
Material and method
1. main agents:
High-Affinity GSH Resin is Nanjing Genscript Biotechnology Co., Ltd.'s product, and IPTG, DTT are
BIOSHARP Products.Other reagent is domestic or Import Analysis is pure.
2. the ultrasonic degradation of expression staphylococcus aureus surface albumen FnBPA engineering bacteria:
The engineering bacteria of the expression Staphylococcus aureus surface protein FnBPA cultivated is poured in Centrifuge Cup, 8000rpm, 4
DEG C, centrifugal 20mins, abandon supernatant, thalline is resuspended in the lysate of original fluid 1/10 volume, and lysate is 20mmol/L PB
PH8.0,10 mmol/L EDTA, 1mmol/L DTT, 5% glycerol, ice-bath ultrasonic breaks bacterium 10mins, 8000rpm, 4 DEG C, centrifugal
20mins, collects supernatant, abandons precipitation.The supernatant collected is for affinitive layer purification.
3. the purification of expression staphylococcus aureus surface albumen FnBPA:
Supernatant solution adds the Resin gel 10ml of counter-balanced High-Affinity GSH, and 4 DEG C combine overnight, loading,
Collection penetrates liquid.With 1 × PBS(pmsf Han 1mM of ten times of bed volumes) washing pillar, then wash with the GSH of 50ml high concentration
De-liquid, eluent is 50 mmol/L Tris-HCl pH8.0,10 mmol/L GSH, in three times eluting destination proteins, is
The staphylococcus aureus surface protein fragments of purification.
Result
The albumen of eluting on High-Affinity GSH Resin post is carried out SDS-PAGE analysis, and result shows, warp
Induction substantially gives expression to FnBPA/GST fusion protein, and expression product is primarily present in supernatant, molecular weight 42kDa, sees Fig. 5.
OD is recorded after eluting280=1.4, calculate concentration 1.2mg/ml, SDS-PAGE Explicit Expression product purity is more than 90%.
The application of the staphylococcus aureus surface albumen FnBPA of purification
The staphylococcus aureus surface albumen FnBPA antigen that purification is obtained, immunity new zealand white rabbit, prepare multi-resistance
Serum.Use indirect elisa method detection antiserum titre, as a result, it was confirmed that the staphylococcus aureus surface albumen that purification obtains
FnBPA antigen has preferable immunogenicity and antigenicity.
Material and method
1. main material:
New zealand white rabbit is purchased from Nanjing Hua Bukang biological product company, and 96 hole elisa plates are that Shenzhen Jin Canhua company produces
Product, complete Freund's adjuvant and incomplete Freund's adjuvant are sigma Products, and Goat Anti-rabbit IgG HRP is Beijing
Bo Aosen Products, TMB nitrite ion is beyotime Products.Other reagent are domestic or Import Analysis is pure.
2. prepared by animal immune and antiserum:
The Healthy female new zealand white rabbit of two about 2kg of picking, auricular vein takes blood about 1ml, before preparation immunity just
Often serum, as negative control.The staphylococcus aureus surface albumen FnBPA that purification is obtained by first immunisation is complete with Freund
Adjuvant 1:1 by volume pushes into Emulsion in syringe, carries out dorsal sc multi-point injection.2w, 4w after initial immunity
The FnBPA obtained by purification and incomplete Freund's adjuvant 1:1 by volume pushes into Emulsion in syringe, carries out dorsal sc many
Point injection.Inject the pure antigen of FnBPA at 6w, take blood examination after 1w and survey serum titer.Carotid artery takes blood, and room temperature places 1h, and 4 DEG C quiet
Put overnight, be centrifuged 30min with 2500g, take supernatant ,-20 DEG C of preservations.
3. the ELISA detection of immune serum:
The staphylococcus aureus surface albumen FnBPA that purification obtains is diluted to 1 g/ml and is coated 96 hole elisa plates, 4 DEG C
Overnight.Washing 3 times with PBST, every hole adds 20% calf serum confining liquid 130 μ l, closes 2h for 37 DEG C.Rabbit anti-serum is opened from 1:1000
Beginning to dilute, negative control dilutes in proportion, 37 DEG C of reaction 1h.After PBST washs 3 times, add the IgG of the goat antirabbit of HRP labelling
(1:5000), 37 DEG C of reaction 30min.After PBST washs 5 times, adding TMB nitrite ion, 37 DEG C of lucifuge colour developing 10min, with 1mol/L
HCl terminates reaction, detects A by microplate reader450Value.Using the antibody greatest dilution of P/N >=2.1 as titer final value.
Result
Using ELISA method to detect sero-fast titer, rabbit anti-serum starts dilution from 1:1000, before taking non-immunity simultaneously
Serum shows as negative control, ELISA result, and after FnBPA pure antigen booster immunization, serum titer is more than 1:400000, negative
Comparison can not be shown in Fig. 6 in conjunction with colour developing, illustrates that the staphylococcus aureus surface albumen FnBPA antigen that purification obtains has preferably
Immunogenicity and antigenicity.
The gene fragment order table of the FnBPA epitope enrichment region of chemosynthesis
<110>Li Yuexi
<120>the surface protein FnBPA partial gene fragments of the staphylococcus aureus of chemosynthesis and expression thereof, should
With
<160> 2
<210> 745
<211> 877
<212> PRT
<213>FnBPA epitope enrichment region
<220>
<223>the surface protein FnBPA fragment of the staphylococcus aureus containing strong antigen epi-position, the 745th amino acids
877th amino acids, totally 133 aminoacid.
<400> 1
Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Glu Asp
5 10 15
Lys Pro Lys Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp
20 25 30
Ser Val Pro Gln Ile His Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu
35 40 45
Glu Asp Thr Glu Lys Asp Lys Pro Lys Tyr Glu His Gly Gly Asn Ile
50 55 60
Ile Asp Ile Asp Phe Asp Ser Val Pro His Ile His Gly Phe Asn Lys
65 70 75 80
His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys Pro Ser Tyr
85 90 95
Gln Phe Gly Gly His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro
100 105 110
Lys Val Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile Glu Glu Asp Thr
115 120 125
Thr Pro Pro Ile Val
130
<210> 2
<211> 402
<212> DNA
<213>artificial sequence
<220>
<221> CDS
<222> (1)...(399)
<223>the brand-new genetic fragment of synthetic, the surface protein FnBPA fragment of coding staphylococcus aureus.
<220>
<221> mis-feature
<222> (400)...(402)
<223>termination codon increased during synthetic gene.
<400> 2
GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT AAG CCG AAG TAT 60
Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Glu Asp Lys Pro Lys Tyr
1 5 10 15 20
GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG CAG ATT CAT GGC CAA 120
Glu Gln Gly Gly Asn Ile Val Asp Ile Asp Phe Asp Ser Val Pro Gln Ile His Gly Gln
25 30 35 40
AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT AAG CCG AAA TAT GAA CAC 180
Asn Lys Gly Asn Gln Ser Phe Glu Glu Asp Thr Glu Lys Asp Lys Pro Lys Tyr Glu His
45 50 55 60
GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG CAT ATC CAC GGC TTC AAC AAG 240
Gly Gly Asn Ile Ile Asp Ile Asp Phe Asp Ser Val Pro His Ile His Gly Phe Asn Lys
65 70 75 80
CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT AAA CCG AGC TAC CAA TTT GGC GGT 300
His Thr Glu Ile Ile Glu Glu Asp Thr Asn Lys Asp Lys Pro Ser Tyr Gln Phe Gly Gly
85 90 95 100
CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG CCG AAA GTT TCC GGT CAG AAC GAA GGC 360
His Asn Ser Val Asp Phe Glu Glu Asp Thr Leu Pro Lys Val Ser Gly Gln Asn Glu Gly
105 110 115 120
CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG CCG ATT GTT TGA 420
Gln Gln Thr Ile Glu Glu Asp Thr Thr Pro Pro Ile Val
125 130
Claims (3)
1. a genetic fragment of the staphylococcus aureus surface albumen FnBPA of chemosynthesis, this genetic fragment coding is containing strong
The staphylococcus aureus surface albumen FnBPA genetic fragment of epitope, the i.e. the 745th amino acids to the 877th amino acids,
Totally 133 aminoacid, the 5' end in this genetic fragment adds BamH I restriction enzyme site (underscore part), adds at 3' end
Termination codon TGA and XhoI restriction enzyme site (underscore part), gene order total length 414bp of chemosynthesis, sequence is as follows:
GGATCC GGT CAG AAC AGC GGT AAC CAG TCG TTT GAA GAA GAC ACG GAA GAA GAT
AAG CCG AAG TAT GAA CAG GGT GGC AAC ATT GTG GAC ATT GAT TTT GAC AGT GTC CCG
CAG ATT CAT GGC CAA AAC AAA GGT AAT CAG TCC TTC GAA GAA GAC ACC GAA AAA GAT
AAG CCG AAA TAT GAA CAC GGC GGT AAC ATT ATC GAT ATT GAC TTT GAT AGC GTT CCG
CAT ATC CAC GGC TTC AAC AAG CAT ACC GAA ATT ATC GAA GAA GAC ACG AAT AAG GAT
AAA CCG AGC TAC CAA TTT GGC GGT CAC AAT TCT GTG GAC TTC GAA GAA GAT ACG CTG
CCG AAA GTT TCC GGT CAG AAC GAA GGC CAG CAG ACG ATT GAA GAA GAC ACG ACG CCG
CCG ATT GTT TGACTCGAG。
2. utilize the genetic fragment of chemosynthesis described in claim 1, use technique for gene engineering to express this gene order and compile
The staphylococcus aureus surface albumen FnBPA genetic fragment of code, the protein fragments that purification is expressed, concrete grammar is as follows:
The structure of staphylococcus aureus surface albumen FnBPA genetic fragment recombiant plasmid:
With the staphylococcus aureus surface albumen FnBPA base of BamHI and XhoI double digestion plasmid pGEX4T-2 and chemosynthesis
Because of fragment, after electrophoresis reclaims, connect with T4 DNA ligase, make staphylococcus aureus surface albumen FnBPA genetic fragment insert
Enter between the BamH I in vector pGEX 4T-2 and XhoI site, consistent with the start codon translation framework on carrier, table
Reaching a fusion protein, 359 aminoacid of total length, this fusion protein N end has merged 226 aminoacid on carrier, and C end comprises
The 745th amino acids to the 877th amino acids in staphylococcus aureus surface albumen FnBPA, full length amino acid sequence is such as
Under:
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg
Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu
Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro
Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile
Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met
Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys
Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met
Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro
Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu
Asp Ala Phe Pro Lys Leu Val Cys Phe Lys
Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr
Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro
Lys Ser Asp Leu Val Pro Arg Gly Ser Gly Gln Asn Ser Gly Asn Gln Ser Phe Glu
Glu Asp Thr Glu Glu Asp Lys Pro Lys Tyr Glu Gln Gly Gly Asn Ile Val Asp Ile
Asp Phe Asp Ser Val Pro Gln Ile His Gly Gln Asn Lys Gly Asn Gln Ser Phe Glu
Glu Asp Thr Glu Lys Asp Lys Pro Lys Tyr Glu His Gly Gly Asn Ile Ile Asp Ile
Asp Phe Asp Ser Val Pro His Ile His Gly Phe Asn Lys His Thr Glu Ile Ile Glu
Glu Asp Thr Asn Lys Asp Lys Pro Ser Tyr Gln Phe Gly Gly His Asn Ser Val Asp
Phe Glu Glu Asp Thr Leu Pro Lys Val Ser Gly Gln Asn Glu Gly Gln Gln Thr Ile
Glu Glu Asp Thr Thr Pro Pro Ile Val
The Screening and Identification of expressed fusion protein engineering bacteria:
By recombinant plasmid transformed e. coli bl21, the coating LB flat board containing 100 μ g/ml ampicillin, put 37 DEG C overnight,
Next day, random picking converted bacterium colony and the comparison bacterium containing plasmid pGEX4T-2, extracted plasmid, and double digestion is verified, cuts out 414bp's
Purpose band；
Plasmid containing exogenous gene carries out DNA sequence analysis, and sequence analysis confirms that recombiant plasmid contains Staphylococcus aureus
Bacterium fnbpa genetic fragment, sequence is the most correct；By the positive transformant containing recombiant plasmid, it is seeded to containing ampicillin 100
In the LB culture medium of μ g/mL, 37 DEG C of shaken cultivation 4h, add IPTG to final concentration 0.2 mmol/L, continue shaken cultivation induction
4h, centrifugal thalline of collecting carries out SDS-PAGE detection, and recon expresses the S. aureus L-forms surface protein that relative molecular weight is 42 kD
FnBPA, expression is 20%, and the comparison bacterium containing empty plasmid pGEX4T-2 is without this protein band；
The purification of expression staphylococcus aureus surface albumen FnBPA:
Being centrifuged by the engineering bacteria of abduction delivering fusion protein, receive bacterium, thalline is resuspended in lysate, and lysate is 20mmol/L PB
PH8.0,10 mmol/L EDTA, 1mmol/L DTT, 5% glycerol, ice-bath ultrasonic breaks bacterium 75 times, centrifugal collects supernatant, and supernatant is molten
Liquid adds the Resin gel 10ml of counter-balanced High-Affinity GSH, and 4 DEG C combine overnight, and loading is collected and penetrated liquid；With
1 × PBS of ten times of bed volumes washs pillar, and then with the GSH eluent of 50ml high concentration, eluent is 50 mmol/L
Tris-HCl pH8.0,10 mmol/L GSH, in three times eluting destination proteins, be the S. aureus L-forms FnBPA albumen flakes of purification
Section.
3. the gene fragment order of the staphylococcus aureus surface albumen FnBPA of the chemosynthesis described in claim 1, it is special
Levy and be, it is also possible to utilize yeast cells, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, system
Standby.
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| Antibody Response to Fibronectin-Binding Adhesin FnbpA in Patients with Staphylococcus aureus Infections;FABRIZIA CASOLINI 等;《INFECTION AND IMMUNITY》;19981231;第66卷(第11期);第5433页摘要，第5440页左栏倒数第1段，第5441页左栏第2段 * |
| Signas,C.等.RecName: Full=Fibronectin-binding protein A;Flags: Precursor，UniProtKB/Swiss-Prot: P14738.1.《GenBank》.2013, * |
| 金黄色葡萄球菌FnbpA、ClfA、Ebps 抗原表位的串联表达及多克隆抗体的制备;高翔 等;《中国畜牧兽医》;20111231;第38卷(第5期);97-100 * |
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Effective date of registration: 20230912 Address after: 210002 No. 293 East Zhongshan Road, Jiangsu, Nanjing Patentee after: EASTERN THEATER DISEASE PREVENTION AND CONTROL CENTER OF PLA Address before: 210002 Military Medical Research Institute, Nanjing military area command, 293 East Zhongshan Road, Nanjing, Jiangsu Patentee before: Li Yuexi |